(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property organization
`‘
`International Bureau
`
`(43) International Publication Date
`7 August 2003 (07.08.2003)
`
`
`
`PCT
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`llllllIllllllllllllllllllllllllllllllllIll!llllllllllllllllllllllllllllllllllll
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`(10) International Publication Number
`W0 03/064604 A2
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`(5]) International Patent Classification":
`
`CIZN
`
`(21) International Application Number:
`
`PCT/USOS/O’JA24
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`(22) International Filing Date: 27 January 200387012003)
`
`(25) Filing Language:
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`(26) Publication Language:
`
`_ English
`
`English
`
`(74) Agent: SHERWOOD, Pamela J.; Bozicevic, Field &
`Francis LLP, 200 Middlefield Road, Suite 200, Menlo Park1
`CA 94025 (US).
`
`(81)
`
`Designated States (hational): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH,
`' GM, HR, I-IU, ID, IL, IN, IS, JP, ICE, KG, KP, KR, KZ, LC,
`LK, LR. LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, M2, NO, NZ, OM, PH, ‘PL, PT, RO, RU, SC. SD, SE,
`SG, SK, SL, TJ, TM, TN, TR, ’l‘T, TZ, UA, UG, US, UZ,
`VC, VN, YU, ZA, ZM, ZW.
`
`(30) Priority Data:
`60/351,919
`
`25 .lanuary 2002 (25.01.2002) US
`
`(84)
`
`(71) Applicant (for all designated States except US): THE
`BOARD OF TRUSTEES OF THE LELAND STAN-
`
`V FORD JUNIOR UNIVERSITY [US/US]; 1705 El
`Camino Real, Palo Alto, CA 94306—1106 (US).
`
`Designated States (regional): ARIEO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG. KZ, MD, RU, TJ, TM),
`European patent,(AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI. FR, GB, GR, HU, IE, IT, .LU, MC, NI... PT, SE, SI,
`SK, TR), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`.
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): PUGLISI, Joseph,
`D. [USIUS]; 773 Mayfield Avenue, Stanford, CA 94305
`(US). BLANCHARD, Scott, c. [US/US] ; 275 Alameda de
`1as Pulgas, Redwood City, CA 94062 (US). GONZALEZ,
`Ruben, L. [US/US]; 818 West A Street #44, Hayward, CA
`94541(US).
`~-
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`'-'
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`'-
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`Published:
`without international search report and to be republished
`upon receipt ofthat report
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`For twooletter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes andA bbreviations ” appearing at the begin—
`‘ning ofeach regular issue ofthe PCT Gazette.
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`llllilllllllllllllllllIllllllllllllllllllllllIlllllllllllllllllllllllllllllllllllll
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`3/064604A2
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`(-
`
`) Title: TRANSLATION PROFILING
`
`(57) Abstract: Surface-bound, translationally competent ribosome complexes are used to generate a translation profile for mRNA,
`which mRNA may be a, single molecular species, or a combination of species, including complex mixtures such as those found in
`the set of mRNAs isolated from a cell or tissue. One or more components of the surface-bound ribosome complex may be labeled
`at specific positionsvto permit analysis of multiple or single molecules for determination of n’bosomal conformational changes and
`translation kinetics. Translation profiles are used as the basis for comparison of an mRNA or set of mRNA species. The translation
`0 profile can be used to determine such characteristics as kinetics of initiation, kinetic of elongation, identity of the polypeptide product,
`and the like. Analysis of translation profiles may be used to determine differential gene expression, optimization of mRNA sequences .
`3
`for expression, screening drug candidates for an effect on translation, etc.
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`PCT{U803102424
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`TRANSLATION PROFILING
`
`BACKGROUND OF THE INVENTION -
`
`Protein synthesis is performed by the ribosome, which in conjunction with many
`exogenous factors translates the genetic code into protein. This process of translation has
`important practical aspects. The ribosome is a target for many clinically important
`
`antibiotics, and tools to monitor the ribosome and translation find use in drug screening.
`Vl'i'ranslation also provides the route from gene to'expressed protein.
`Translation of the mRNA genetic code into protein is the final step in genetic
`information transfer. While current methods of gene expression analysis can determine the
`
`“10'
`
`. cellular levels of individual mRNAs, these must be assumed to correlate with the final
`" amounts of the encoded proteins. However,
`in many cases translation of mRNA by the
`
`~15
`
`ribosome has been shown to be dependent on the sequence and structure of the mRNA.
`Therefore, assessment of an expression profile by looking solely at mRNA levels ignores
`the subtleties and. regulation of translationby the ribosome. Often, translation initiation is
`the rate limiting step in protein synthesis; in' addition, different mRNAs are translated at
`different rates through differences in the elongation rate of protein synthesis. Methods of
`
`screening for translation of mRNAs could provide an important means 'of evaluating gene
`expression.
`.
`The ribosome is also an important target for a wide variety of antibiotics. Many of
`them, such asstreptomycin and tetracycline, were of great clinical importance when they
`
`f
`
`were first discovered, but unfortunately strains of bacteria with resistance to these drugs
`
`have become commonplace, limiting their effectiveness. At the same time, many other
`antibiotics targeting the ribosome have insufficient specificity toward bacterial (as opposed
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`25
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`to eukaryotic) ribosomes, and hence are too toxic for routine clinicai use in humans. With
`
`the emergence ‘of new multi—drug resistant strains of bacteria, there is a real need to
`
`understand details of how these antibiotics interact with the ribosome, and for screening
`methods to assess new drug candidates.
`,
`V.
`Many of the ribosome-directed antibiotics target rRNA, which forms critical functional
`
`'30
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`sites on the ribosome. The antibiotics are thus both powerful mechanistic tools to dissect
`individual steps of protein synthesis, and lead compounds for the development of novel
`
`therapeutic agents. The ribosome and translation are important targets for therapeutic.
`
`intervention, not only for treatment of infectious disease. but also treatment of human
`diseases that involve protein expression.
`'
`
`'35
`'
`
`-
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`' The rich structure! information on the ribosome lies in stark contrast to knowledge‘of
`_
`its dynamics. ' Systems that permit the analysis of translation areofégreat interest for
`,
`‘. synthetic and screening methods. .
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`Relevant Publications
`The analysis of single molecule fluorescence is disclosed in, for example Ha et at
`' 1999) Proc Natl Acad Sci U S A 96(3): 893-8; Ha et al. (1999) Proc Natl Acad Sci U S A
`96(16): 9077-82;- Weiss (1999) Science 283(5408): 1676—83; and Zhuang et a1. (2000)
`
`I
`
`Science288(5473): 2048—51.
`The use of ribosome display is discussed, for example, by Amstutz et al. (2001) Curr
`:. Opin Biotechnol 200112(4):400-5; and by Hanes et aIf (2000) Methods Enzymol
`
`. 2000;328:4041-30.
`'
`‘ Ribosome structure and function are reviewed by Puglisi at al. (2000) Nat Struct Biol
`-7(10):855-61; and Green and Puglisi (1999) Nat Struct Biol 6(11):999-1003. Eukaryotic
`ribosome function is reviewed, for example, by Lafontaine at a]. (2001) Nat Rev Mol Cell
`
`~
`
`10.
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`Biol 2(7):514-2o.
`
`SUMMARY OF THE iNVENTlON
`
`3.15
`
`‘
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`~
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`Compositions and methods are provided for analysis of protein synthesis utilizing
`‘- surface-bound, translationally competent riboSome complexes. The spatial localization of
`
`'
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`this translational system permits both large scale translation procedures, and arrays of
`highly parallel translation reactions. These methods find use in the analysis of expressed
`mRNAs for their ability to produce protein; for screening individual mRNA templates for the
`'- ability to be translated into protein, for screening biological agents for their ability to
`
`'- ---20
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`" enhance or interfere with translation, and the like.
`ln one embodiment of the invention, the surface translation system is used to
`generate a translation profile for mRNA, which mRNA may be a single molecular species, or
`I a combination of species,
`including complex mixtures such as those found in the set of
`mRNAs isolated from a cell or tissue. Translation profiles can be used as the basis for
`comparison of an mRNA or set of mRNA Species. The translation profile can be used to
`determine such charaCteristics as kinetics of initiation, kinetic of elongation, identity of the
`polypeptide product, and the like. Analysis of translation profiles may be used to determine
`differential gene expression, optimization of mRNA sequences for expression, screening
`
`. drug candidates for an effect on translation, etc.
`One or more components of the surface—bound ribosome complex may be labeled at
`- specific positions to permit analysis of multiple or single molecules for determination of.
`ribosomal conformational changesand translation kinetics. The surface bound system of
`
`25
`
`'30
`
`the present invention allows the detection of' an effect on translation from altering the
`~ translational environment, where the environment may include exogenous agents, e.g. drug
`
`'.35
`
`-' candidates; mRNA sequence changes; salt concentration; pH, the presence of factors; and
`
`the like. Such methods are useful in qualitative, quantitative, and competitive assays.
`.
`g
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`UETAlLED DESCRIPTION OF THE INVENTION
`
`.
`
`.Flexible multiplex screening assays are provided for the screening and biological
`activity classification of biologically active agents and protein coding sequences; A surface .
`translation system is used to generate a translation profile for mRNA, which mRNA may be
`a single molecular species, or a combination of species, including complex mixtures such
`‘ as those found in the set of mRNAs isolated from a cell or tissue. Translation profiles can
`be used as the basis for comparison of an mRNA or set of mRNA species. The translation
`profile can be used to determine such characteristics as kinetics of initiation, kinetic of
`elongation, identity of the polypeptide product, and the like. Analysis of translation profiles
`can be used to determine differential gene expression, optimization of mRNA sequences for
`expression, screening druggcandldates for an effect on translation, etc. The measurement
`of translation kinetics provides highly complementary information to other methods of gene
`’ expression analysis, e.g. quantitation and differentiation of mRNA populations.
`Translationally competent ribosome complexes are immobilized ‘on a solid surface
`The site of attachment is selected so as to avoid steric interference with translation, and
`may be accomplished through the use of a specific binding partner to ribosomal RNAs;
`mRNA; ribosomal proteins, and other polynucleotide or polypeptide components. A spatial
`.array of immobilized ribosomes may be produced on a planar substrate, microbeads, on
`fiber optics; and the like.
`
`One or more components of the surface—bound ribdsome complex may be labeled at
`specific positions to permit analysis of multiple or single molecules for determination of
`__ translation kinetics. Ribosomal RNAs, including mRNA and tRNA; ribosomal proteins; and
`.. other factors and agents involved in translation may be labeled at specific positions, and
`arrays of immobilized ribosomes may comprise a panel of different labels and positions of
`labels.
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`1O
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`.15.
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`20
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`-
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`:25
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`g
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`Detection of the label can then be used to monitor translation kinetics, such as the
`initiation and elongation rats of protein synthesis. Single molecule analysis can detect rare
`.events that are not obsenled in bulk, ensemble-averaged measurements, and allow
`heterogeneity in the system to be sorted and characterized, allowing the analysis of overall
`translation rates for different mRNAs bound to the surface. For multistep processes such
`as translation, single molecule analysis eliminates the requirement for synchronization of
`large numbers of molecules. Distance scales probed by methods such as fluorescence
`
`resonance energy transfer (FRET) are on the order of about 20—80A, which permits
`determination of translation kinetics. To performsingle—molecule analysis of a biomolecular
`4. system, the molecules are specifically localized on a derivatized quartz surface, where the
`. attachment to the surface allows spatial localization of the particle to the Optical limit of the
`microscope without'Impairing its function
`
`30
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`m some embodiments of the invention, the polypeptide product is screened for
`,- function, presence of epitopes, binding. etc, by localizing the polypeptide product at or near
`the site of the surface bound ribosome, for example by independently binding the
`polypeptide to the surface, by maintaining the polypeptide bound to the ribosome, and the
`like.
`.
`'
`
`TRANSLATION PROFlLE
`
`1‘0-
`
`To generate a translation profile, a test sample comprising an mRNA or set of
`mRNAs of interest is combined with a translationally competent ribosome~ complex. The
`ribosome complex may be bound to a surface prior to combination with the mRNA, or may
`be immobilized after compiexing with the mRNA. At
`least one component of the
`mRNA/ribosome complex will comprise a detectable label, and preferably at least two
`' components are separately labeled with fluorochromes that form a donor/acceptor pair for
`FRET. Translation kinetics,
`i.e.
`the rate of initiation of translation, and/or translation
`elongation, and/or
`translation termination can be determined through fluorescence
`spectroscopy of such label(s).
`in one embodiment of the invention.'singie' molecule
`fluorescence is used to determine the translation kinetics. For example, FRET analysis of
`the interaction between a labeled ribosome and separately labeled mRNA can be used to
`determine the translation kinetics of a single mRNA molecule.
`.
`Further information may be included in a translation profile by the addition of
`translation kinetics from samples comprising variation in sequence, mRNA composition,
`and/orreaction conditions. Reactions conditions may include the addition of exogenous
`. agents that affect translation, e.g. antibiotics; by variation in ionicity, temperature, biological
`._ factors, etc. Sequence changes can be made to the mRNA to determine, for example, the
`, effect of codon usage, three~dimensional structure and the like on translation. Data points
`from two or more combinations of sequence and reaction condition can be compared, for
`example to a similarly obtained control sample which may be a positive or a negative
`.‘ control. The comparison may be a subtraction of the two values, ratio of the two, etc.
`Comparison can also be made against
`libraries of compounds, where the translation
`kinetics in the presence of one agent is compared to the translation kinetics in the presence
`.I of another agent, which may be unrelated, or may be related or analogous compounds.
`The results can be entered into a data processor to provide a translation profile
`.dataset. Algorithms are used for the comparison and analysis of translation profiles
`obtained under different conditions. The effect of sequence, factors and/or agents is read
`out by determining changes in translation kinetics in the translation profile. The translation
`profile will include the results from the test sample, and may also include one or more of the
`other samples as described above. _A database of translation profiles can be compiled from
`4
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`15
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`20.
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`25
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`30
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`- sets of experiments, for example, a database can contain translation profiles obtained from
`
`~.a panel of different mRNA sequences, with multiple different changes in reaction conditions,
`where each change can be a series of related compdunds, or oompoUnds representing
`different classes of molecules.
`
`Mathematical systems can be used to compare translation profiles, and to provide
`
`quantitative measures of similarities and differences between them. For example, the
`translation profiles in the database can be analyzed by pattern recognition algorithms or
`clustering methods, eg. hierarchical or k—means clustering, etc, that Use statistical analysis
`‘to quantify relatedness.
`These methods can be modified by weighting, employing
`classification strategies, etc. to optimize the ability of a translation profile to discriminate
`different functional effects. -
`'
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`10
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`. MRNATEST SAMPLES
`
`. The mRNA for analysis can be prepared according to conventional methods,
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`15
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`including isolation from cells where the cells may be prokaryote or eukaryote, e.g. freshly
`
`.
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`-
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`isolated biological samples taken from an organism, cultured cells, genetically modified
`
`cells, etc; or the mRNA can be prepared by in vitro transcription reactions,
`
`in vitro
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`synthesis, and the like. The mRNA can comprise a single sequence, which can be a
`naturally existing sequence or a genetically modified sequence. Alternatively, complex
`, mixtures of mRNA can be evaluated, e.g. when isolated from a biological sample.
`
`20
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`25'
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`30_
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`A large number of public resources are available as a source of genetic sequences,
`
`'-e.g. for human, other mammalian, bacterial, plant, protozoan, and animal sequences. A
`substantial portion of the human genome is sequenced, and can be accessed through
`public databases such as Genbank Resources include the uni-gene set, as well as
`genomic sequences.
`cDNA clones corresponding to many human gene sequences are
`available from the lMAGE consortium. The international lMAGE Consortium laboratories
`
`develop and array cDNA clones for worldwide use. The clones are commercially available,
`
`for example from Genome Systems, Inc., St. Louis, MO.
`in some cases the mRNA will be hybridized, particularly at the 5’ end, with a labeled
`oligonucleotide.
`For example, eukaryotic mRNA can be hybridized to a labeled poly—
`thymidine or poly-uridine probe. Suitable hybridization conditions are well known to those of
`
`:skill in the art and reviewed in Molecular Cloning: A Laboratory Manual (Sambrook et at,
`
`Cold Spring Harbor Laboratory Press, New York, 1989). Labeling of the oligonucleotide
`
`probe is performed by conventional methods known to those of skill in the art.
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`METHODS OF SCREENING MRNA TEST SAMPLES
`
`Various methods are utilized to generate a translation profile from an mRNA sample.
`For example, a labeled oligonucleotide may be hybridized downstream on the mRNA of
`choice, and the hybridized mRNA then combined with a surface bound ribosome complex,
`where the ribosome complex comprises a label thatIs a complementary donor/acceptor to
`the oligonucleotide label.
`.Translation is initiated by buffer exchange with an translation
`. extract, e.g. wheat germ, E. coli S'lOO extract, etc. Translation elongation is measured as
`
`.
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`.10'
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`. appearance of. a FRET signal as the labeled ribosome approaches the labeled
`oligonucleotide. The dye label on the ribosome can be attached to the 308 subunit, near
`:where the 3'end of the mRNA exits from the ribosome, e.g. the cleft near ribosomal protein
`85 is the leading edge of the translating ribosome. Thus, labeling sites on the ribosome
`side would include a beak or H16 label, as discussed in more detail below. An alternate
`
`labeling approach utilizes reconstituted 308 particles with labeled 35 protein; a number of
`single-cysteine mutants of 85 have been derivatized and successfully incorporated into 308
`subunits.
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`15
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`in one embodiment of the invention, mRNAs are isolated from cells and mRNAs
`
`undergoing translation initiation or elongation are coupled to the encoded protein
`undergoing synthesis via the ribosome. This is done using commercially available, small
`molecule antibiotic drugs, e.g. aminoglycosldes, that reversibly lock down and arrest the
`
`‘ 1.20
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`translation apparatus thereby linking genotype and phenotype. mRNAs arrested in this
`
`manner are then isolated from the cell and hybridized to a DNA array comprising
`__ oligonucleotides complementary to downstream portions of the different mRNAs. The
`
`translation kinetics can be determined using FRET.
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`25
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`
`in another embodiment, labeled DNA oligonucleotides of from about 6 to about 20,
`usually about 8 tom nucleotides are pre-hybridized to mRNAIn the test sample, Where the
`site for hybridization is immediately downstream from the initiation codon. An initiation
`__ complex with the hybridized mRNA—DNA complex is assembled on a solid surface, and
`translation initiated by buffer exchange with an translation extract, e.g. wheat germ, E. coli
`$100 extract, etc. The labeled oligonucleotide is displaced by the ribosome when its
`
`.~ leading edge hits the duplex, about 15 his from the 5’-position of the A—site oodon.
`Elongation rates. are measured from the lag time until-loss of fluorescence. Similarly, two
`labeled oligonucleotides that each comprise one member of a donor acceptor fluorochrome
`
`,
`
`. pair may be hybridized successively downstream of the start codon. Translation is initiated,
`_ e.g. by buffer exchange with a suitable extract, and sequential loss of fluorescence from the
`
`'35,
`
`fluorochromes is measured.
`
`In another embodiment of the invention, translation is initiated'In the presence of a
`labeled oiigonucleotide complementary to the region of mRNA occluded by the ribosomeIn
`6.
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`the initiation complex. When sufficient polypeptide elongation has occurred to move the
`ribosome downstream of
`the initiation site,
`the ,mRNA is
`free to hybridize the
`_ oligonucleotide, thereby providing a signal for FRET.
`
`I"
`
`I.
`
`An alternative method utilizes mRNA‘that comprises an epitope for which a high
`,
`affinity antibody is available. Numerous such epitopes are known in the art, 9.9.
`the
`. sequence encoding the amino acid EQKLlSEEDL, which is the epitope for high-affinity
`, binding by anti—myc antibody. The epitope will be exposed to the antibody upon its
`. emersion from the 508 subunit exit tunnel, which protects about ‘40-50 amino acids.
`
`_ Binding of labeled antibody will lead to localization, of the label, which means at least about
`
`: 40-50 amino acids have been synthesized. The epitope tag can be incorporated into any
`coding sequence of interest, and may be positioned at varying sites throughout the coding
`__ sequence.
`From the time lag before localization of fluorescence as a function of tag
`position, translation rates can be estimated. As an alternative to an epitope tag, peptide
`sequences that form fluorescent arsenate complexes can be inserted into the coding
`sequence. Translation of such modified mRNA is performed in the presence of the labeling
`arsenic compound.
`
`CANDIDATE AGENT TEST SAMPLES
`
`interest are biologically active agents that encompass
`Candidate agents of
`_ numerous chemical classes, primarily organic molecules, which may include organometallic
`molecules,
`inorganic molecules, genetic sequences, etc. An important aspect of the
`invention is to evaluate candidate drugs for an effect on translation. Candidate agents
`comprise functional groups necessary for structural interaction with proteins, particularly
`hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl
`‘ group, frequently at least two of the functional chemical groups. The candidate agents often
`comprise cyclical carbon or heterocycllc structures and/or aromatic or polyaromatic
`structures substituted with one or more of the above functional groups. Candidate agents
`'.'are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty
`acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations-thereof.
`Test compounds include all of the classes of molecules described above, and may
`further compriselsamples of unknown content. Of interest are complex mixtures of naturally
`. occurring compounds derived from natural sources such as plants. While many samples
`will comprise compounds in solution, solid samples that canrbe dissolved in a suitable
`solvent may also be assayed. Samples of interest include environmental samples, e.g.
`ground water, sea water, mining waste, etc; biological samples, e.g. lysates prepared from
`crops, tissue samples, etc; manufacturing samples, e.g. time course during preparation of
`pharmaceuticals; as well as libraries ofgcompounds prepared, for analysis; andthe like.
`7
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`- Samples ofinterest include compounds being assessed for potential therapeutic value, i.e.
`drug candidates.
`I
`g
`The term samples also includes the fluids described above to which additional
`components have been added, for example components that affect the ionic strength, pH,
`_ total protein concentration, etc.
`ln addition, the samples may be treated to achieve at least
`
`partial fractionation or concentration. Biological samples may be stored if care is taken to
`
`. reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof.
`
`,The volume of sample used is sufficient to allow for measurable detection, usually from
`
`about 0.1 pl to 1 ml of a biological sample is sufficient.
`
`10 _-
`
`Compounds,
`
`including candidate agents, are obtained from a wide variety of
`
`. sources including libraries of synthetic or natural compounds. For example, numerous,
`
`means are avaiiable for random and directed synthesis of a wide variety of organic
`
`including expression of randomized oligonucleotides
`including biomolecules,
`compounds,
`and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial,
`fungal, plant and animal extracts are available or readily produced. Additionally, natural or
`
`'15
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`,
`
`_ synthetically produced libraries and compounds are readily modified through conventional
`
`"chemical, physical and biochemical means, and may be used to produce combinatorial
`‘ libraries. Known pharmacological agents may be subjected to directed or random chemical
`
`modifications, such as acylation, alkylation, esterification, amidification, etc,
`
`to produce
`
`1120
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`. structural analogs.
`
`'
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`.
`
`ANTlBlOTICS
`
`A number of clinically important drugs interfere with protein translation, and find use
`
`in the generation of
`
`translation profiles, as well ' as providing target moiecules for
`
`modification and development of new therapeutic entities. These compounds find use in
`binding mRNA to ribosome complexes, for ribosome labeling purposes, for investigation of
`, conformation and kinetics in translation, and in drug development.
`
`Compounds of interest include aminoglycosides,.which inhibit protein synthesis by
`irreversibly binding to 308 ribosomal subunit. Furthermore, these antibiotics interfere with
`
`human immunodeficiency virus (HIV)
`replication by disrupting essential RNA-protein
`contacts.
`Aminoglycosides currently in
`clinical use include amikacin, gentamicin,
`
`25~
`
`-
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`'
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`' 13.0.
`
`~- kanamycin,
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`netilmycin,
`
`neomycin B,
`
`paromomycin,
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`streptomycin and tobramycin.
`
`- Hygromycin B is active against both prokaryotic and eukaryotic cells, and differs in structure
`
`from other aminoglycosides by having a dual ester linkage between two of its three sugar
`amoieties resulting in a fourth. 5-membered ring. The drug works primarily by inhibiting the
`
`.35 .
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`translocation step of elongation and, to a lesser. extent, causes misreading of mRNA.
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`ln
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`‘ 8
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`BNSDOCID: <W
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`03054604A2_l_>
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`

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`W0 03/064604
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`PCT/USO3/02424
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`eukaryotes, the antibiotic affects EF—2-mediated translocation of A site bound tRNA to the P
`
`. site, accompanied byan increase in the affinity of the A site for aminoacyl-tRNA.
`Aminoglycoside antibiotics are multiply charged compounds of high'flexibility. The-
`
`,positive charges are attracted to the negatively charged RNA backbone. The flexibility of
`
`.. the aminoglycosides facilitates accommodation into a binding pocket within internal loops of
`
`-- .RNA helices or into ribozyme cores for making specific contacts. The majority of these
`
`. antibiotics are composed of amino sugars linked to a 2-deoxystreptamine ring. The
`
`- conserved elements among aminoglycosides are rings I and ii and, within ring ii, the amino
`
`groups at positions 1 and 3. These elements are essential for binding to the decoding site
`
`.10'
`
`.lof the 16S rRNA. The Zedeoxystreptamine ring is substituted, most commonly, at positions
`
`.4 and 5, as in the neomycin class, or at positions 4 and 6, as in the kanamycin and
`gentamicin classes.
`I
`
`The tetracyclines inhibit protein synthesis by binding to 308 ribosomal subunit and
`
`- blocking binding of aminoacyl transfer-RNA.
`
`it appears likely, however, that the initial
`
`_15
`
`-' binding of a ternarytcomplex'of EF-Tu with tRNA to the A site and the process of decoding
`
`.-are not affected since ribosome-dependent GTP hydrolysis by EF-Tu is unaffected by
`:-tetracycline. Tcs have no apparent effect on the binding of tRNA to the P site except during
`
`factor-dependent initiation. Consistent with the inhibition of tRNA binding to the A site during
`
`- translation, Tcs also prevent binding of both release factors RF-1 and 2 during termination,
`
`regardless of the stop codon. Tetracyclines currently in clinical use include demeclocycline,
`
`doxycycline, methacycline, minocycline, oxytetracycline and tetracycline.
`
`The macrolides inhibit protein synthesis by binding to 508 ribosomal subunits,
`
`,
`
`inhibiting translocation‘ of peptidase chain and inhibiting polypeptide synthesis. This group
`
`2 includes azithromycin, clarithromycin, dirithromycin and erythromycin. The lincosamide
`
`._antibiotics, e.g., clindamycin and iincomycin, interfere with transpeptidation and early chain
`
`20
`
`'25
`
`, termination.
`
`Linezoiid inhibits the first step of protein synthesis by binding to i—met—t~RNA—mRNA—
`
`- 30$ ribosome subunit. Evernimicin (Evn), ‘an oligosaccharide antibiotic, interacts with the
`
`large ribosomal subunit and inhibits bacterial protein synthesis by interacting with a specific
`
`.'.30
`
`_ set of nucleotides in the loops of hairpins 89' and 91 of 23S rRNA in bacterial and archaeai
`
`-, ribosomes.
`
`Pactamycin (Pct) was isolated from Streptomyces pactum as a potential new human
`
`antitumor drug, but is in fact a potent inhibitor of translation in all three kingdoms, eukarya,
`
`‘35..
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`bacteria, and archaea.
`For this reason, the drug is expected to interact with highly
`conserved regions of 16S RNA, both structurally and with respect to sequence.
`in bacteria,
`" Pct inhibits the initiation step of translation. Binding of the drug prevents release of initiation
`
`0-
`
`factors from the 308 initiationcomplex, which in turn prevents thefonnation of functional
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`9
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`BNSDOClD: <wo
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`whammy)
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`wo 03/064604
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`PCT/USO3/02424
`
`708 ribosomes. The antibiotic interferes with factor and GTP—dependent binding of tRNA to
`
`the ribosome! P site during initiation, but factor-free initiation does not seem tobe affected.
`
`METHODS OF SCREENING CANDIDATE AGENTS
`
`Samples comprising candidate agent are screened for their effect on translation, by
`
`- combining the candidate agent with a surface bound translation complex comprising at least
`
`, one mRNA species capable of , translation by the system. Agents are screened for
`
`: biological activity by adding the agent to at least one. and in some cases a plurality, of
`
`combinations of translation complexes; "The change in ribosome conformation and/or
`
`10
`
`translation kinetics in responseto .the agent is measured, desirably normalized, and the
`
`resulting translation profile may then be evaluated by comparison to reference translation
`
`~ profiles. The reference translation profiles may include rea'douts in the presence and
`absence of other agents, 9:9. antibiotics with known action, positive controls, etc. Agents of
`
`. interest for analysis include any biologically active molecule with the potential tomodulate
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`7:15 . =-
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`translation.
`
`.
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`The agents are conveniently added in solution, or readily soluble form,
`
`to the
`
`'.medium of the surface bound ribosome complex. The agents may be added in a flow—
`
`through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of
`
`the compound, singly or incrementally, to an othenNise static solution. Preferred agent
`
`'9"-=.--20..'
`
`formulations do not include additional components, such as preservatives, thatmay have a
`
`. significant effect on the overall formulation.
`
`A plurality of assays may be run in parallel with different agent concentrations to
`
`obtain a differential response to